Pancreatic Cancer Genes

ABSTRACT

The present invention provides the art with the DNA coding sequences of polynucleotides that are up-or-down-regulated in cancer and dysplasia. These polynucleotides and encoded proteins or polypeptides can be used in the diagnosis or identification of cancer and dysplasia. Inhibitors of the up-regulated polynucleotides and proteins can decrease the abnormality of cancer and dysplasia. Enhancing the expression of down-regulated polynucleotides or introducing down-regulated proteins to cells can decrease the growth and/or abnormal characteristics of cancer and dysplasia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of (and claims the benefit ofpriority under 35 U.S.C 120) U.S. patent application Ser. No.10/351,953, filed Jan. 24, 2003, which is a continuation of U.S. patentapplication Ser. No. 09/773,459, filed Jan. 31, 2001, now U.S. Pat. No.6,664,054, which is a divisional of U.S. patent application Ser. No.09/337,171, filed Jun. 21, 1999, now U.S. Pat. No. 6,262,249, whichclaims priority from provisional application Ser. No. 60/118,570, filedFeb. 3, 1999, and provisional application Ser. No. 60/090,391, filedJun. 23, 1998. This application also claims the benefit of PCTApplication Number PCT/1999/14036 filed Jun. 22, 1999. The disclosure ofthe prior applications are considered part of (and is incorporated byreference in) the disclosure of this application

TECHNICAL AREA OF THE INVENTION

The invention relates to the area of diagnosis and treatment ofpancreatic cancer and dysplasia. More specifically, it relates topolynucleotides which are differentially regulated in pancreatic cancerand dysplasia.

BACKGROUND OF THE INVENTION

Pancreatic cancer is the fifth leading cause of cancer death in theUnited States. According to the American Cancer Society, approximately28,000 people will die of pancreatic cancer in the United States in1998. A high risk of developing pancreatic cancer, without acorresponding increase in the risk of developing other cancers, may bepassed along in some families. Pancreatic cancer is most likely causedby an accumulation of mutations in specific cancer-causing genes.Pancreatic cancer is very aggressive and chemotherapeutic agents whichmay be active against other malignancies do not work effectively whenused for pancreatic cancer.

The majority of cells in the pancreas are in the exocrine glands, whichproduce pancreatic enzymes, and in the ducts that carry the pancreaticenzymes to the bile duct and to the small intestine. Cancers of theexocrine cells of the pancreas are usually adenocarcinomas. Pancreaticadenocarcinomas usually begin in the ducts of the pancreas, but maysometimes develop from the acinar cells. About 95% of cancers of thepancreas are adenocarcinomas. Less common cancers of the exocrinepancreas include adenosquamous carcinomas, squamous cell carcinomas, andgiant cell carcinomas.

Because pancreatic cancer is an aggressive cancer with very highmortality, there is a need in the art for genes that are up-ordown-regulated in tumor progression Such genes are useful fortherapeutic purposes and for diagnosis of pancreatic as well as othercancers.

SUMMARY OF THE INVENTION

The invention provides isolated polynucleotides comprising codingregions or portions of genes whose expression is mis-regulated in cancerand dysplasia.

The invention also provides isolated proteins and protein fragmentswhose expression is mis-regulated in cancer and dysplasia.

The invention further provides an antibody preparation whichspecifically binds to a polypeptide the expression of which ismis-regulated in cancer and dysplasia.

The invention provides a method for diagnosing cancer and dysplasia.

The invention still further provides therapeutic compositions useful fortreating cancer and dysplasia.

These and other objects of the invention are provided by one or more ofthe embodiments described below. One embodiment of the inventionprovides isolated polynucleotides comprising at least twelve contiguousnucleotides selected from the group of polynucleotide sequences as shownin SEQ ID NOS:1-15.

Another embodiment of the invention provides isolated polypeptidescomprising at least six contiguous amino acids encoded by apolynucleotide selected from the group consisting of the polynucleotidesequences as shown in SEQ ID NOS:1-15.

Even another embodiment of the invention provides an antibodypreparation which specifically binds to a polypeptide comprising atleast six contiguous amino acids encoded by a polynucleotide selectedfrom the group of polynucleotide sequences as shown in SEQ ID NOS:1-15.

Yet another embodiment of the invention provides isolated nucleotideprobes consisting of a sequence selected from the group consisting ofthe polynucleotide sequences shown in SEQ ID NOS:1-15.

Still another embodiment of the invention provides a method ofdiagnosing cancer. The amount of a polypeptide expressed from apolynucleotide having a sequence as shown in SEQ ID NO: 12 in a testsample of tissue of a human suspected of being cancerous is determined.The amount of said polypeptide is also determined in a human tissuewhich is normal. The determined amounts are then compared. A test samplewhich contains less of the polypeptide than the normal tissue isidentified as cancerous.

A further embodiment of the invention provides an additional method ofdiagnosing cancer. The amount of specific mRNA molecules in a testsample of tissue suspected of being cancerous and in a human tissuewhich is normal are determined. The mRNA molecules to be measured arecomplementary to the minus strand of a double-stranded polynucleotidesequence. The double-stranded polynucleotide sequence is shown in SEQ IDN0.12. The determined amounts of mRNA molecules are compared. A testsample of tissue which contains less of the mRNA molecules than thenormal tissue is identified as cancerous.

Another embodiment of the invention provides a therapeutic compositionuseful for reducing the growth rate of cancer cells The composition iscomprised of a polynucleotide comprising all or a portion of anucleotide sequence which is operably linked to a promoter sequence anda pharmaceutically acceptable carrier. The polynucleotide comprising allor a portion of a nucleotide sequence comprises at least 18 contiguousnucleotides. The nucleotide sequence is shown in SEQ D NO: 12.

Yet another embodiment of the invention provides a therapeuticcomposition useful for reducing the growth rate of cancer cells. Thecomposition is comprised of a polypeptide comprising all or a portion ofan amino acid sequence expressed from a polynucleotide sequence and apharmaceutically acceptable carrier. The polynucleotide sequence isshown in SEQ ID NO: 12.

Another embodiment of the invention provides a method of diagnosingdysplasia and cancer. The amount of a polypeptide expressed from apolynucleotide having at least one of a sequence selected from the groupconsisting of the polynucleotide sequences shown in SEQ ID NOs:2, 5, and15 in a test sample of tissue suspected of being dysplastic or cancerousis determined. The amount of the polypeptide is also determined in ahuman tissue which is normal. The determined amounts are compared. Atest sample of human tissue which contains more of at least onepolypeptide than the normal tissue is identified as being dysplastic orcancerous.

A further embodiment of the invention provides another method ofdiagnosing dysplasia. The amount of a polypeptide expressed from apolynucleotide having a sequence selected from the group consisting ofthe polynucleotide sequences shown in SEQ ID NOS: 1, 3-4, 6-11, and13-14 is determined in a test sample of tissue suspected of beingdysplastic. The amount of said polypeptide is also determined in a humantissue which is normal The two amounts are then compared. A test sampleof human tissue which contains more of said polypeptide than the normaltissue is identified as being dysplastic.

Another embodiment of the invention provides an additional method ofdiagnosing cancer.

The amount of a polypeptide expressed from a polynucleotide having asequence selected from the group consisting of the polynucleotidesequences shown in SEQ ID NOs: 2,5, and 15, is determined in a testsample of tissue suspected of containing cancer, and in a human tissuewhich is normal. The amount of a polypeptide expressed from apolynucleotide having a sequence selected from the group consisting ofthe polynucleotide sequences shown in SEQ ID NOs: 1, 3-4, 6-11, and13-14 is also determined in the test sample, and in the normal tissueThe determined amounts of said polypeptides are then compared. A testsample of tissue which contains more of the polypeptide expressed from apolynucleotide having a sequence selected from the group consisting ofthe polynucleotide sequences shown in SEQ ID NOs:2, 5, and 15, ascompared to the normal tissue, and which contains substantially the sameamount of a polypeptide expressed from a polynucleotide selected fromthe group as shown in SEQ ID NOs:1, 3-4, 6-11, and 13-14, as compared tothe normal tissue, is identified as cancerous.

Even another embodiment of the invention provides a method of diagnosingdysplasia and cancer The amount of specific mRNA molecules is determinedin a test sample of tissue suspected of being dysplastic or cancerousand in a human tissue which is normal The mRNA molecules measured arecomplementary to the minus strand of a double-stranded polynucleotidesequence The double-stranded polynucleotide sequence is selected fromthe group of polynucleotides as shown in SEQ ID NOs.2, 5, and 15 Thedetermined amounts of mRNA molecules are compared. A test sample ofhuman tissue which contains more of the mRNA molecules than the normaltissue is identified as being dysplastic or cancerous.

Yet another embodiment of the invention provides a method of diagnosingdysplasia. The amounts of specific mRNA molecules in a test sample ofhuman tissue suspected of being dysplastic and in a human tissue whichis normal are determined. The mRNA molecules are complementary to theminus strand of a double-stranded polynucleotide sequence. Thedouble-stranded polynucleotide sequence is selected from the group ofpolynucleotides as shown in SEQ ID NOS:1, 3-4, 6-11, and 13-14. Thedetermined amounts of mRNA molecules are then compared. A test sample ofhuman tissue which contains more of the mRNA molecules than the normaltissue is identified as being dysplastic.

Still another embodiment of the invention provides a method ofdiagnosing cancer. The amounts of a first set of specific mRNA moleculesin a test sample of tissue of a human suspected of being cancerous andin a human tissue which is normal are determined. The mRNA molecules arecomplementary to the minus strand of a double- stranded polynucleotidesequence. The double-stranded polynucleotide sequence is selected fromthe group of polynucleotide sequences as shown in SEQ ID NOs: 1, 3-4,6-11, and 13-14. In addition, the amounts of a second set of specificmRNA molecules in a test sample of tissue of a human suspected of beingcancerous and in a human tissue which is normal are determined. The mRNAmolecules are complementary to the minus strand of a double-strandedpolynucleotide sequence. The double-stranded polynucleotide sequence isselected from the group of polynucleotide sequences as shown in SEQ IDNOs: 2, 5, and 15. The determined amounts of the first and second setsof mRNA molecules are compared. A test sample of human tissue whichcontains more of the second set of mRNA molecules than the normaltissue, and which contains substantially the same amount of the firstset of mRNA molecules, as compared to the normal tissue, is identifiedas cancerous.

Yet another embodiment of the invention provides a therapeuticcomposition useful for decreasing the amount of translation of an mRNAmolecule in a cell. The composition comprises an antisensepolynucleotide complementary to the plus strand of a double-strandedpolynucleotide. The double-stranded polynucleotide is selected from thegroup consisting of polynucleotides comprising a nucleotide sequence asshown in SEQ ID NOs:1-11, and 13-15, wherein said antisensepolynucleotide binds to an mRNA molecule. The composition also includesa pharmaceutically acceptable carrier.

A further embodiment of the invention provides a therapeutic compositionuseful for reducing the expression of a polypeptide. The compositioncomprises an antibody which specifically binds to a polypeptideexpressed from a polynucleotide selected from the group consisting ofpolynucleotides comprising a nucleotide sequence as shown in SEQ IDNOs:1-11 and 13-15. The composition also includes a pharmaceuticallyacceptable carrier.

Another embodiment of the invention provides a therapeutic compositionuseful for reducing the translation from an mRNA molecule Thecomposition comprises a ribozyme which binds to an mRNA molecule,wherein a portion of said ribozyme is complementary to the plus strandof a double-stranded polynucleotide. The polynucleotide is selected fromthe group consisting of the polynucleotides comprising a sequence asshown in SEQ ID NOs: 1-11, and 13-15. The composition also comprises apharmaceutically acceptable carrier.

The present invention provides the art with useful polynucleotides whichrepresent expressed sequences of genes. Expression of the genes ismis-regulated in cancer. The invention also provides the art withdiagnostic methods based on the over- and under-expression of the genesand the polypeptides encoded by the genes in cancer and dysplastic cellsInhibitors of the over-expressed polynucleotides and polypeptides can beused to reduce the growth of cancer cells and dysplastic cells. Thepolynucleotides and polypeptides which are under-expressed in cancer anddysplasia can be delivered therapeutically to reduce the abnormalcharacteristics of cancer cells and dysplastic cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Polynucleotides that are mis-regulated in cancer and dysplasia aredisclosed. The mis-regulated polynucleotide sequences are shown in SEQ DNOS: 1-15. The polynucleotides are mis-regulated as follows:

SEQ ID NO: 12 is down-regulated in cancer;

SEQ ID NOs:2, 5, and 15 are up-regulated in cancer and dysplasia; and

SEQ ID NOS:1, 3-4, 6-11, and 13-14 are up-regulated in dysplasia only.

Polynucleotides that are differentially regulated in cancer or dysplasiaor both can be useful in the diagnosis and treatment of these diseases.Dysplasia is an atypical proliferation of epithelial or mesenchymalcells that may represent an early stage of cancer; however, dysplasiadoes not necessarily progress to cancer. Epithelial dysplasia results inthe loss of normal orientation of one epithelial cell to another,accompanied by alterations in cellular and nuclear size and shape.Cancer is a proliferation of malignant cells that are no longer undernormal physiologic control.

The subgenomic polynucleotides of the invention contain less than , awhole chromosome and are preferably intron-free. The subgenomicpolynucleotides of the invention can be isolated and purified free fromother nucleotide sequences by standard nucleic acid purificationtechniques, for example, using PCR, cloning, and/or restriction enzymesand probes to isolate fragments comprising the encoding sequences.Subgenomic polynucleotides of the invention can include all or acontiguous portion of a gene coding region. In one embodiment, anisolated and purified subgenomic polynucleotide of the inventioncomprises at least 10, 11, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60,70, 74, 80, 90, 100, 125, 150, 154, 175, 200, 250, 300, or 350contiguous nucleotides selected from the polynucleotide sequences asshown in SEQ ID NOs:1-15. In a preferred embodiment, the polynucleotidemolecules comprise a contiguous sequence of at least twelve nucleotidesselected from the group consisting of the polynucleotides shown in SEQID NOs:1-15.

An open reading frame is a region of DNA that consists exclusively oftriplets that represent amino acids The open reading frame of thepolynucleotide sequences of the invention can be determined by examiningall three possible reading frames in both directions. If a reading framecontains termination codons it cannot be read into protein and is notconsidered an open reading frame. Usually, no more than one of the sixpossible frames is open in any single stretch of DNA. An extensive openreading frame is unlikely to exist by chance because of the lack ofselective pressure to prevent the accumulation of nonsense codons.Therefore, the identification of a lengthy open reading frame is takento be prima facie evidence that the sequence is translated into proteinin that frame. Lewin, ed,. 1990, Genes IV, Cell Press, Cambridge, Mass.

Subgenomic polynucleotides of the invention can be used, inter alia, toproduce proteins or polypeptides, as probes for the detection of mRNA ofthe invention in samples or extracts of human cells, to generateadditional copies of the polynucleotides, and to generate ribozymes orantisense oligonucleotides. The subgenomic polynucleotides can also beused as single stranded DNA probes or as triple strand formingoligonucleotides The probes can be used to determine the presence orabsence of the polynucleotide sequences as shown in SEQ ID NOs:1-15 orvariants thereof in a sample.

The sequence of a nucleic acid comprising at least 15 contiguousnucleotides of at least any one of SEQ ID NO:1-15, preferably the entiresequence of at least any one of SEQ ID NO: 1-15, is not limited and canbe any sequence of A, T, G, and/or C (for DNA) and A, U, ,G, and/or C(for RNA) or modified bases thereof, including inosine andpseudouridine. The choice of sequence will depend on the desiredfunction and can be dictated by coding regions desired, the intron-likeregions desired, and the regulatory regions desired.

Where the entire sequence of any one of SEQ ID NO:1-15 is within thenucleic acid, the nucleic acid obtained is referred to herein as apolynucleotide comprising the sequence of any one of SEQ ID NO: 1-15.

Both secreted and membrane-bound polypeptides of the present inventionare of interest. For example, levels of secreted polypeptides can beassayed conveniently in body fluids, such as blood and urine.Membrane-bound polypeptides are useful for constructing vaccine antigensor inducing an immune response. Such antigens would comprise all or partof the extracellular region of the membrane-bound polypeptides.

Because both secreted and membrane-bound polypeptides comprise afragment of contiguous hydrophobic amino acids, hydrophobicitypredicting algorithms can be used to identify such polypeptides.

A signal sequence is usually encoded by both secreted and membrane-boundpolypeptide genes to direct a polypeptide to the surface of the cell.The signal sequence usually comprises a stretch of hydrophobic residues.Such signal sequences can fold into helical structures.

Membrane-bound polypeptides typically comprise at least onetransmembrane region that possesses a stretch of hydrophobic amino acidsthat can transverse the membrane. Some transmembrane regions alsoexhibit a helical structure.

Hydrophobic fragments within a polypeptide can be identified by usingcomputer algorithms. Such algorithms include Hopp & Woods, Proc. Natl.Acad. Sci. USA 78:3824-3828 (1981); Kyte & Doolittle, J. Mol. Biol.157:105-132 (1982); and RAOAR algorithm, Degli Esposti el al., Eur. J.Biochem. 190:207-219 (1990).

Another method of identifying secreted and membrane-bound polypeptidesis to translate the present polynucleotides, SEQ ID NO: 1-15, in all sixframes and determine if at least 8 contiguous hydrophobic amino acidsare present. Those translated polypeptides with at least 8; moretypically, 10; even more typically, 12 contiguous hydrophobic aminoacids are considered to be either a putative secreted or membrane boundpolypeptide. Hydrophobic amino acids include alanine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, threonine, tryptophan, tyrosine, and valine.

The polypeptides of the invention include those encoded by the disclosedpolynucleotides. These polypeptides can also be encoded by nucleic acidsthat, by virtue of the degeneracy of the genetic code, are not identicalin sequence to the disclosed polynucleotides. Thus, the inventionincludes within its scope nucleic acids comprising polynucleotidesencoding a protein or polypeptide expressed by a polynucleotide havingthe sequence of any one of SEQ ID NO: 1-15. Also within the scope of theinvention are variants; variants of polypeptides include mutants,fragments, and fusions. Mutants can include amino acid substitutions.additions or deletions. The amino acid substitutions can be conservativeamino acid substitutions or substitutions to eliminate non-essentialamino acids, such as to alter a glycosylation site, a phosphorylationsite or an acetylation site, or to minimize misfolding by substitutionor deletion of one or more cysteine residues that are not necessary forfunction. Conservative amino acid substitutions are those that preservethe general charge, hydrophobicity/hydrophilicity, and/or steric bulk ofthe amino acid substituted For example, substitutions between thefollowing groups are conservative: Gly/Ala, VaVIleLeu, AspIGlu, Lys/Arg,Asn/Gln, Ser/Cys,Thr, and Phe/Trp/Tyr.

Cysteine-depleted muteins are variants within the scope of theinvention. These variants can be constructed according to methodsdisclosed in U.S. Pat. No. 4,959,314, “Cysteine-Depleted Muteins ofBiologically Active Proteins.” The patent discloses how to substituteother amino acids for cysteines, and how to determine biologicalactivity and effect of the substitution. Such methods are suitable forproteins according to this invention that have cysteine residuessuitable for such substitutions, for example to eliminate disulfide bondformation.

The protein variants described herein are encoded by polynucleotidesthat are within the scope of the invention. The genetic code can be usedto select the appropriate codons to construct the correspondingvariants.

The invention encompasses polynucleotide sequences having at least 65%sequence identity to any one of SEQ ID NOS: 1-15 as determined by theSmith-Waterman homology search algorithm as implemented in MSPRCHprogram (Oxford Molecular) using an affine gap search with the followingsearch parameters gap open penalty of 12, and gap extension penalty of1.

Polynucleotide probes comprising at least 12 contiguous nucleotidesselected from the nucleotide sequence of a polynucleotide of SEQ IDNO:1-15 are used for a variety of purposes, including identification ofhuman chromosomes and determining transcription levels.

The nucleotide probes are labeled, for example, with a radioactive,fluorescent, biotinylated, or chemiluminescent label, and detected bywell known methods appropriate for the particular label selectedProtocols for hybridizing nucleotide probes to preparations of metaphasechromosomes are also well known in the art A nucleotide probe willhybridize specifically to nucleotide sequences in the chromosomepreparations which are complementary to the nucleotide sequence of theprobe. A probe that hybridizes specifically to a polynucleotide shouldprovide a detection signal at least 5-. lo-, or 20-fold higher than thebackground hybridization provided with other unrelated sequences.

Polynucleotides of the present invention are used to identify achromosome on which the corresponding gene resides. Using fluorescencein situ ‘hybridization (FISH) on normal metaphase spreads, comparativegenomic hybridization allows total genome assessment of changes inrelative copy number of DNA sequences. See Schwartz and Samad, CurrentOpinions in Biotechnology (1994) 8:70-74; Kallioniemi et al., Seminarsin Cancer Biology (1993) 4:41-46; Valdes and Tagle, Methods in MolecularBiology (1997) 68:1 , Boultwood, ed., Human Press, Totowa, N.J.

Preparations of human metaphase chromosomes are prepared using standardcytogenetic techniques from human primary tissues or cell lines.Nucleotide probes comprising at least 12 contiguous nucleotides selectedfrom the nucleotide sequence of SEQ ID NOS: 1-15 are used to identifythe corresponding chromosome. The nucleotide probes are labeled,, forexample, with a radioactive, fluorescent, biotinylated, orchemiluminescent label, and detected by well known methods appropriatefor the particular label selected. Protocols for hybridizing nucleotideprobes to preparations of nietaphase chromosomes are also well known inthe art. A nucleotide probe will hybridize specifically to nucleotidesequences in the chromosome preparations that are complementary to thenucleotide sequence of the probe A probe that hybridizes specifically toa polynucleotide-related gene provides a detection signal at least 5-,10-, 25 or 20-fold higher than the background hybridization providedwith non-polynucleotide coding sequences.

Polynucleotides are mapped to particular chromosomes using, for example,radiation hybrids or chromosome-specific hybrid panels. See Leach elal., Advances in, Genetics, (1995) 33:63-99, Walter et al., NatureGenetics (1994) 7:22-28; Walter and Goodfellow, Trends in Genetics.(1992) 9:352. Such mapping can be useful in identifying the function ofthe polynucleotide-related gene by its proximity to other genes withknown function. Function can also be assigned to the related gene whenparticular syndromes or diseases map to the same chromosome.

A polynucleotide will be useful in forensics, genetic analysis, mapping,and diagnostic applications if the corresponding region of a gene ispolymorphic in the human population. A particular polymorphic form ofthe polynucleotide may be used to either identify a sample as derivingfrom a suspect or rule out the possibility that the sample derives fromthe suspect. Any means for detecting a polymorphism in a gene are used,including but not limited to electrophoresis of protein polymorphicvariants, differential sensitivity to restriction enzyme cleavage, andhybridization to an allele-specific probe.

Any naturally occurring variants of the nucleotide sequences whichencode variants thereof are within the scope of this invention. Allelicvariants of subgenomic polynucleotides of the invention can occur andcan be identified by hybridization of putative allelic variants withnucleotide sequences disclosed herein under stringent conditions. Forexample, by using the following wash conditions—2×SCC, 0.1% SDS, roomtemperature twice, 30 minutes each, then 2×SCC, 0.1% SDS, 50 ″C once, 30minutes; then 2×SCC, room temperature twice, 10 minutes each—allelicvariants of the polynucleotides of the invention can be identified whichcontain at most about 25-30% base pair mismatches. More preferably,allelic variants contain 15-25% base pair mismatches, even morepreferably 5-15%, or 2-5%, or 1-2% base pair mismatches.

Amplification by the polymerase chain reaction (PCR) can be used toobtain the polynucleotides of the invention, using either genomic DNA orcDNA as a template. The polynucleotides of the invention may also beobtained using reverse transcriptase and mRNA molecules that arecomplementary to the minus strand of a double-stranded sequence whereinsaid double-stranded sequence is selected from the group ofpolynucleotides comprising a sequence as shown in SEQ ID NOS: 1-15.Using the polynucleotide sequences disclosed herein, subgenomicpolynucleotide molecules of the invention can also be made using thetechniques of synthetic chemistry.

Probes specific to the polynucleotides of the invention may be generatedusing the polynucleotide sequences disclosed in SEQ ID NOS: 1-15. Theprobes are preferably at least 12, 14, 16, 18, 20, 22, 24, or 25nucleotides in length and can be less than 2, 1, 0.5, 0.1, or 0.05 kb inlength. The probes can be synthesized chemically or can be generatedfrom longer polynucleotides using restriction enzymes. The probes can belabeled, for example, with a radioactive, biotinylated, or fluorescenttag.

Subgenomic polynucleotides of the invention can be propagated in vectorsand cell lines using techniques well known in the art. Expressionsystems in bacteria include those described in Chang et al., Nature(1978) 275: 615; Goeddel et al., Nature (1979) 281:544; Goeddel et al.,Nucleic Acids Res. (1980) 8:4057; EP 36,776; U.S. Pat. No. 4,551,433;deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80:21-25; andSiebenlist et al., Cell (1980) 20:269.

Expression systems in yeast include those described in Hinnen et al.,Proc. Natl. Acad. Sci. USA (1978) 75:1929; Ito et al., J. Bacteriol.(1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142, Kunze etal. J. Basic Microbiol. (1985) 25:141; Gleeson et al., J. Gen.Microbiol. (1986) 132:3459, Roggenkamp et al., Mol. Gen. Genet. (1986)202:302; Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt et.al., J. Bacteriol. (1983) 154:737, Van den Berg et al., Bio Technology(1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141; Cregg etal., Mol. Cell. Biol. (1985) 5:3376; U.S. Pat. No. 4,837,148, U.S. Pat.No. 4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al.,Curr. Genet. (1985) 10:380; Gaillardin et al., Curr. Genet. (1985)10:49; Ballance et al., Biochem. Biophys. Res. Commun. (1983)112:284-289; Tilburn et al., Gene (1983) 26:205-221, Yelton et al.,Proc. Natl. Acad. Sci. USA (1984) 81:1470-1474; Kelly and Hynes. EMBO J.(1985) 4:475479; EP 244,234; and WO 91/00357.

Expression of the subgenomic polynucleotides of the invention in insectscan be accomplished as described in U.S. Pat. No. 4,745,051, Friesen ela/. (1986) “The Regulation of Baculovirus Gene Expression” in: THEMOLECULAR BIOLOGY OF BACULOVIRUSES (W. Doerfler, ed.); EP 127,339; EP155,476; Vlak et al., J. Gen. Virol. (1988) 69:765-776; Miller et al.,Am. Rev. Microbiol. (1988) 42:177; Carbonell et al., Gene (1988) 73:409;Maeda et al., Nature (1985) 315:592-594; Lebacq-Verheyden et al., Mol.Cell. Biol. (1988) 8:3129, Smith et al., Proc. Natl. Acad. Sci. USA(1985) 82:8404; Miyajima et al., Gene (1987) 58:273; and Martin et al.,DNA (1988) 7:99. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts are described inLuckow et al., Bio/Technology (1988) 6:47-55; Miller et al., in GENETICENGINEERING (Setlow, J. K. et al. eds.), Vol. 8 (Plenum Publishing,1986), pp. 277-279; and Maeda et al., Nature,( 1985) 315:592-594.

Mammalian expression of the subgenomic polynucleotides of the inventioncan be accomplished as described in Dijkema et al., EMBO J (1985) 4:76;Gorman et al., Proc. Natl. Acad. Sci. USA (1982) 79:6777; Boshart etal., Cell (1985) 41:521; and U.S. Pat. No. 4,399,216. Other features ofmammalian expression can be facilitated as described in Ham and Wallace,Meth. Enz. (1979) 58:44; Barnes and Sato, Anal. Biochem. (1980) 102:255,U.S. Pat. No. 4,767,704; U.S. Pat. No. 4,657,866; U.S. Pat. No.4,927,762; U.S. Pat. No. 4,560,655; WO 901103430; WO 87/00195, and U.S.RE 30,985.

The subgenomic polynucleotides of the invention can be on linear orcircular molecules. They can be on autonomously replicating molecules(vectors) or on molecules without replication sequences. They can beregulated by their own or by other regulatory sequences, as is known inthe art The subgenomic polynucleotides of the invention can beintroduced into suitable host cells using a variety of techniques whichare available in the art, such as transferrin-polycation-mediated DNAtransfer, transfection with naked or encapsulated nucleic acids,liposome-mediated DNA transfer, intracellular transportation ofDNA-coated latex beads, protoplast fusion, viral infection,electroporation, gene gun, and calcium phosphate-mediated transfection.

The invention provides a method of detecting expression of apolynucleotide in, for example. a biological sample, which can beuseful, inter alia, for diagnosing cancer or dysplasia. The basis forthis method is the discovery that the polynucleotide sequence(s) asshown in:

SEQ ID NO: 12 is down-regulated in cancer;

SEQ ID NOs: 2. 5, and 15 are up-regulated in cancer and dysplasia; and

SEQ ID NOS: 1. 3-4, 6- 11, and 13-14 are up-regulated in dysplasia only.

In patients who have been diagnosed with pancreatic dysplasia or cancer,the detection of levels of the expression products of the polynucleotidesequences of the invention, either mRNA or protein, can be used todiagnose or prognose a disorder, to monitor treatment of the disorder,or to screen agents which affect the disorder.

The expression products of the polynucleotide sequences of theinvention, either mRNA or proteins, can be detected in a body sample fordiagnosis or prognosis. The body sample can be, for example, a solidtissue or a fluid sample. The patient from whom the body sample isobtained can be healthy or can already be identified as having acondition in which altered expression of a protein of the invention isimplicated.

In one embodiment, the body sample is assayed for the level of a proteinexpressed from a polynucleotide sequence of the invention. The proteincould be detected by, for example, antibodies to the proteins. Theantibodies can be labeled, for example, with a radioactive, fluorescent,biotinylated, or enzymatic tag and detected directly, or can be detectedusing indirect immunochemical methods, using a labeled secondaryantibody. The presence of the protein can be assayed, for example, intissue sections by immunocytochemistry, or in lysates, using Westernblotting, as is known in the art.

The level of the protein in a tissue sample suspected of being cancerousor dysplastic is compared with the level of the protein in a normaltissue. A higher level of the polypeptides expressed from polynucleotidesequences as shown in SEQ ID NOS: 1, 3-4, 6-11, and 13-14 in the suspecttissue, as compared to the normal tissue, indicates the presence ofdysplastic cells in the suspect tissue. A higher level of thepolypeptides expressed from polynucleotide sequences as shown in SEQ IDNOs: 2, 5, and 15 in the suspect tissue, as compared to the normaltissue, indicates the presence dysplastic cells or cancerous cells orboth in the suspect tissue. A lower level of the polypeptide expressedfrom the polynucleotide sequence as shown in SEQ ID NO: 12 in thesuspect tissue, as compared to the normal tissue, indicates the presenceof cancerous cells in the suspect tissue.

Additionally, a differentiation between cancer or dysplasia in apatient's diagnosis can be made. The expression of a polynucleotidesequence of the invention that is up-regulated in dysplastic cells only( i.e. SEQ ID NOS: 1, 3-4, 6-11, and 13-14) and the expression of apolynucleotide that is up-regulated in both dysplastic cells andcancerous cells (i.e., SEQ ID NOS:2, 5, and 15) can be used to screen apatient's tissues. If examination of a patient's tissues reveals thatthere is no up-regulation of a polynucleotide sequence that isup-regulated in dysplastic cells only (i.e., SEQ ID NOS:1, 3-4, 6-11,and 13-14), and that there is up-regulation of a polynucleotide sequencethat is up-regulated in both cancerous cells and dysplastic cells (i.e.SEQ ID NOs:2, 5, and 15), then the patient is diagnosed with cancer.

Alternatively, the presence of mRNA expressed from the polynucleotidesequences of the invention in two tissues can be compared. mRNA can bedetected, for example, by in situ hybridization in tissue sections, byreverse transcriptase-PCR, or in Northern blots containing poly A+ mRNA.One of skill in the art can readily determine differences in the size oramount of mRNA transcripts between two tissues, using Northern blots andnucleotide probes. For example, the level of mRNA of the invention in atissue sample suspected of being cancerous or dysplastic is comparedwith the expression of the mRNA in a normal tissue. Any methods known inthe art for determining the amounts of specific mRNAs can be used.

A higher level of mRNA expressed from polynucleotide sequences as shownin SEQ ID NOS: 1, 3-4, 6-11, and 13-14 in the suspect tissue, ascompared to the normal tissue, indicates the presence dysplastic cellsin the suspect tissue. A higher level of mRNA expressed from thepolynucleotide sequences as shown in SEQ ID NOs:2, 5, and 15 in thesuspect tissue, as compared to the normal tissue, indicates the presencedysplastic cells or cancerous cells or both in the suspect tissue. Alower level of the mRNA expressed from the polynucleotide sequence asshown in SEQ ID NO: 12 in the suspect tissue, as compared to the, normaltissue, indicates the presence of cancerous cells in the suspect tissue.Any combinations of these sequences can be used to determine adiagnosis.

Optionally, the level of a particular expression product of apolynucleotide sequence of the invention in a body sample can bequantitated. Quantitation can be accomplished, for example, by comparingthe level of expression product detected in the body sample with theamounts of product present in a standard curve. A comparison can be madevisually or using a technique such as densitometry, with or withoutcomputerized assistance Alternative methods can be used, for exampleELISA, western blot, immunoprecipitation, radioimmunoassay, etc. Anymethod known in the art for detecting and quantitating a particularprotein can be used.

Reagents specific for the polynucleotides arid polypeptides of theinvention, such as antibodies and nucleotide probes, can be supplied ina kit for detecting the presence of an expression product in abiological sample. The kit can also contain buffers or labelingcomponents, as well as instructions for using the reagents to detect andquantify expression products in the biological sample.

Polynucleotide expression in a cell can be increased or decreased, asdesired. Polynucleotide expression can be altered for therapeuticpurposes, as described below, or can be used to identify and study therole of therapeutic agents in cancer and other diseases.

Decreasing the expression of genes containing sequences selected fromthe group consisting of the sequences as shown in SEQ ID NOs:1, 3-4,6-11, and 13-14 is useful, for example, as a therapeutic for alteringthe abnormal characteristics of dysplastic cells. Decreasing theexpression of polynucleotide sequences selected from the groupconsisting of the sequences as shown in SEQ ID NOs: 2, 5, and 15 isuseful, for example, as a therapeutic agent for decreasing the growthrate of dysplastic and cancer cells.

Expression of the polynucleotide sequences of the invention can bealtered using an antisense oligonucleotide sequence Therapeuticcompositions for decreasing gene expression comprise an expressionconstruct containing polynucleotides encoding all or a portion of apolynucleotide sequence selected from the group consisting of SEQ IDNOS:1-11, and 13-15 Within the expression construct, the polynucleotidesegment is orientated in the antisense direction and is locateddownstream from a promoter. Transcription of the polynucleotide segmentinitiates at the promoter.

Preferably, the antisense oligonucleotide sequence is at least tennucleotides in length, but longer sequences of at least 11, 12, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 74, 80, 90, 100, 125, 150, 162, 175,200, 250, 300, or 350 contiguous nucleic acids can also be used.Antisense oligonucleotide molecules can be provided in a DNA constructand introduced into cells whose division is to be decreased, asdescribed above. A more complete description of gene transfer vectors,especially retroviral vectors is contained in U.S. Ser. No. 08/869,309,which is incorporated herein by reference.

The antisense oligonucleotides can be composed of deoxyribonucleotides,ribonucleotides, or a combination of both. Oligonucleotides can besynthesized manually or by an automated synthesizer, by covalentlylinking the 5′ end of one nucleotide with the 3′ end of anothernucleotide with phosphodiester or non-phosphodiester internucleotidelinkages such as alkylphosphonates, phosphorothioates,phosphorodithioates, alkylphosphonothioates, alkylphosphonates,phosphoramidates, phosphate esters, carbamates, acetamidate,carboxymethyl esters, carbonates, and phosphate triesters. See Brown,1994, Meth. Mol. Biol. 20:1-8; Sonveaux, 1994, Meth. Mol. Biol. 26:1-72;Uhlmann et al., 1990, Chem. Rev. 90:543-583.

Although precise complementarity is not required for successful duplexformation between an antisense molecule and the complementary codingsequence of a gene, antisense molecules with no more than one mismatchare preferred. One skilled in the art can easily use the calculatedmelting point of an antisense-sense pair to determine the degree ofmismatch which will be tolerated between a particular antisenseoligonucleotide and a particular coding sequence of the selected gene.

The antisense oligonucleotides of the invention can be modified withoutaffecting their ability to hybridize to a polynucleotide coding sequenceof the present invention. These modifications can be internal or at oneor both ends of the antisense molecule. For example, internucleosidephosphate linkages can be modified by adding cholesteryl or diaminemoieties with varying numbers of carbon residues between the aminogroups and terminal ribose. Modified bases or sugars or both, such asarabinose instead of ribose, or a 3′, 5′-substituted oligonucleotide inwhich the 3′ hydroxyl group or the 5′ phosphate group are substituted,can also be employed in a modified antisense oligonucleotide Thesemodified oligonucleotides can be prepared by methods well known in theart. Agrawal et al., 1992, Trends Biotechnol. 10:152-158; Uhlmann etal., 1990, Chem. Rev. 90:543-584, Uhlmann et al., 1987, Tetrahedron.Lett. 215:3539-3542.

Expression of the polynucleotides of the invention can also be decreasedby delivering polyclonal, monoclonal, or single chain antibodies thatspecifically bind to polypeptides expressed from the polynucleotidesequences as shown in SEQ ID NOS:1-11 and 13-15. Antibodies specific tothese proteins bind to the protein and prevent the protein fromfunctioning in the cell. Blocking protein expression or function isuseful for preventing, reducing the effects of, or curing cancer anddysplasia.

In one embodiment of the invention, expression of the polynucleotidesselected from the group consisting of the polynucleotide sequences shownin SEQ ID 15 NOS. 1-11, and 13-15 are decreased using a ribozyme, an RNAmolecule with catalytic activity. See, e.g., Cech, 1987, Science236:1532-1539, Cech, 1990, Ann. Rev. Biochem. 59:543-568, Cech, 1992,Curr. Opinion Struct. Biol. 2:605-609; Couture and Stinchcomb, 1996,Trends Genet. 12:.510-515. Ribozymes an be used to inhibit gene functionby cleaving an RNA sequence, as is known in the art (e.g., Haseloff etal., U.S. Pat. No. 5,641,673).

The coding sequence of a polynucleotide of the invention can be used togenerate a ribozyme which will specifically bind to RNA transcribed fromsaid polynucleotide. Methods of designing and constructing ribozymeswhich can cleave other RNA molecules in trans a highly sequence specificmanner have been developed and described in the art (see Haseloff, J. etal.(1988), Nature 334:585-591). For example, the cleavage activity ofribozymes can be targeted to specific RNAs by engineering a discrete“hybridization” region into the ribozyme. The hybridization regioncontains a sequence complementary to the target RNA and thusspecifically hybridizes with the target (see, for example, Gerlach, W.L. et al., EP 321,201). Longer complementary sequences can be used toincrease the afinity of the hybridization sequence for the target. Thehybridizing and cleavage regions of the ribozyme of the invention can beintegrally related; thus, upon hybridizing to the target RNA through thecomplementary regions, the catalytic region of the ribozyme can cleavethe target.

Ribozymes of the invention can be introduced into cells as part of a DNAconstruct, as is known in the art. The DNA construct can also includetranscriptional regulatory elements, such as a promoter element, anenhancer or UAS element, and a transcriptional terminator signal, forcontrolling the transcription of the ribozyme in the cells.

Mechanical methods, such , as microinjection, liposome-mediatedtransfection, electroporation, gene gun, or calcium phosphateprecipitation, can be used to introduce the ribozyme-containing DNAconstruct into cells whose division it is desired to decrease, asdescribed above. Alternatively. if it is desired that the DNA constructbe stably retained by the cells, the DNA construct can be supplied on aplasmid and maintained as a separate element or integrated into thegenome of the cells,, as is known in the art.

As taught in Haseloff et al., U.S. Pat. No. 5,641,673, the ribozymes ofthe invention can be engineered so that their expression will occur inresponse to factors which induce expression of a polynucleotides of theinvention. The ribozyme can also be engineered to provide an additionallevel of regulation, so that destruction of RNA occurs only when boththe ribozyme and the corresponding gene are induced in the cells.

Preferably, the mechanism used to decrease expression of thepolynucleotides of the invention, whether antisense nucleotide sequence,antibody, or ribozyme decreases expression of the polynucleotide by 50%,60%, 70%, or 80%. Most preferably, expression of the polynucleotide isdecreased by 90%, 95%, 99%, or 100%. The effectiveness of the mechanismchosen to alter expression of the polynucleotide can be assessed usingmethods well known in the art, such as hybridization of nucleotideprobes to mRNA of the polynucleotide, quantitative RT-PCR, or detectionof a protein using specific antibodies of the invention.

Increased expression of a polynucleotide is useful to decrease thegrowth rate of cancer cells where the particular polynucleotide isdown-regulated in cancer cells, such as the polynucleotide sequence asshown in SEQ ID NO:12. Therapeutic compositions for increasingpolynucleotide expression comprise an expression construct containingall or a portion of the polynucleotide sequence as shown in SEQ ID NO:12. Within an expression construct, the polynucleotide segment isoriented in the sense direction and is located downstream from thepromoter. Transcription of the polynucleotide segment initiates at thepromoter. The expression construct can be introduced into cells alongwith a pharmaceutically acceptable carrier to decrease the growth rateof cancer cells or ameliorate other abnormal characteristics. Expressionof the polynucleotide sequence can be monitored by detecting productionof mRNA which hybridizes to the delivered polynucleotide or by detectingprotein encoded by the delivered polynucleotide.

Proteins that are expressed from the polynucleotide sequences of theinvention can be produced recombinantly in prokaryotic or eukaryotichost cells, such as bacteria, yeast, insect, or mammalian cells, usingexpression vectors known in the art. Enzymes can be used to generateless than full length polypeptides by enzymatic proteolysis offull-length proteins of the invention. Alternatively, syntheticchemistry methods, such as solid-phase peptide synthesis, can be used tosynthesize the proteins and polypeptides.

Species homologs of human subgenomic polynucleotides or the encodedpolypeptides can be identified by making suitable probes or primers andscreening cDNA expression libraries from other species, such as mice,monkeys, yeast, or bacteria. Mammalian homologs are preferred, however.

Proteins or polypeptides expressed from the polynucleotide sequences asshown in SEQ ID NO: 1-15 can be isolated and purified from human cellsthat express the proteins. The proteins can be obtained substantiallyfree from other human proteins by standard protein purification methods,such as size exclusion chromatography, ion exchange chromatography,ammonium sulfate fractionation, affinity chromatography, or preparativegel electrophoresis.

Proteins or polypeptides expressed from the polynucleotides of theinvention can also be used in a fusion protein, for example, as animmunogen. The fusion protein comprises two protein segments. The firstprotein segment consists of at least six, eight, ten, twelve, fifteen,twenty or thirty contiguous amino acids of a polypeptide sequenceexpressed from a polynucleotide sequence as shown in SEQ ID NOS:1-15.The first protein segment is fused to a second protein segment by meansof a peptide bond. The second protein segment can be a full-lengthprotein or a fragment of a protein. Techniques for making fusionproteins, either recombinantly or by covalently linking two proteinsegments, are well known in the art.

The second protein or protein fragment of a fusion protein can bederived from another type of protein or a similar protein. The secondprotein or protein fragment can be labeled with a detectable marker,such as a radioactive or fluorescent tag, or can be an enzyme that willgenerate a detectable product Enzymes suitable for this purpose, such asp-galactosidase, are well-known in the art. A fusion protein can beused, for example, to target the proteins of the invention orpolypeptides to a particular location in a cell or tissue. in variousassays, such as the yeast two-hybrid technique, or as an immunogen.

The proteins or polypeptides expressed from the polynucleotides of theinvention can be used for generating antibodies The antibodies can beused, inter alia, to detect and quantitate expression of the cognateprotein. Proteins or polypeptides expressed from the polynucleotides ofthe invention comprising at least six, eight, ten, twelve, fifteen,twenty or thirty consecutive amino acids can be used as immunogens. Theproteins or polypeptides can be used to obtain a preparation ofantibodies which specifically bind to a protein or polypeptide of theinvention. The antibodies can be polyclonal or monoclonal Techniques forraising both polyclonal and monoclonal antibodies are well known in theart.

Single chain antibodies can also be constructed. Single chain antibodieswhich specifically bind to a protein or polypeptide expressed from thepolynucleotides of the invention can be isolated, for example, fromsingle-chain immunoglobulin display libraries, as are known in the art.The library is “panned” against a protein or polypeptide, and a numberof single chain antibodies which bind different epitopes of thepolypeptide with high-affinity can be isolated. Hayashi et al., 1995,Gene 160:129-30. Such libraries are known and available to those in theart. The antibodies can also be constructed using the polymerase chainreaction (PCR), using hybridoma cDNA as a template. Thirion et al.,1996, Eur. J. Cancer Prev. 5:507-11.

The single chain antibody can be mono- or bi-specific, and can bebivalent or tetravalent. Construction of tetravalent bispecific singlechain antibodies is taught in Coloma and Morrison, 1997, Nat.Biotechnol. 15:159-63 Construction of bivalent bispecific single chainantibodies is taught in Mallender and Voss, 1994, J. Biol. Chem.269:199-206.

A nucleotide sequence encoding the single chain antibody can then beconstructed using manual or automated nucleotide synthesis, cloned intoDNA expression vectors using standard recombinant DNA methodologies, andintroduced into cells which express the selected gene, as describedbelow. Alternatively, the antibodies can be produced directly usingfilamentous phage technology Verhaar et al., 1995, Int. J. Cancer61:497-501; Nicholls et al., 1993. J. Immunol. Meth. 165:81-91.

The antibodies bind specifically to the epitopes of the proteins orpolypeptides expressed from the polynucleotides of the invention. In apreferred embodiment, the epitopes are not present on other humanproteins. Typically a minimum number of contiguous amino acids to encodean epitope is 6, 8, or 10. However, more can be used, for example, atleast 15, 25, or 50, especially to form epitopes which involvenon-contiguous residues or particular conformations.

Antibodies that bind specifically to the proteins or polypeptidesinclude those that bind to full-length proteins or polypeptides Specificbinding antibodies do not detect other proteins on Western blots ofhuman cells, or provide a signal at least ten-fold lower than the signalprovided by the target protein of the invention. Antibodies which havesuch specificity can be obtained by routine screening. In a preferredembodiment of the invention, the antibodies immunoprecipitate theproteins or polypeptides expressed from the polynucleotideos theinvention from cell extracts or solution. Additionally, the antibodiescan react with proteins or polypeptides expressed from thepolynucleotides of the invention in tissue sections or on Western blotsof polyacrylamide gels. Preferably the antibodies do not exhibitnonspecific cross-reactivity with other human proteins on Western blotsor in immunocytochemical assays.

Techniques for purifying antibodies to the proteins or polypeptidesexpressed from the polynucleotides of the invention are available in theart. In a preferred embodiment, the antibodies are passed over a columnto which a particular protein or polypeptide expressed from thepolynucleotides of the invention is bound. The bound antibodies are theneluted, for example, with a buffer having a high salt concentration.

Therapeutic compositions of the invention also comprise apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well known to those in the art. Such carriers include, butare not limited to, large, slowly metabolized macromolecule, such asproteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and inactive virusparticles. Pharmaceutically acceptable salts can also be used in thecomposition, for example, mineral salts such as hydrochlorides,hydrobromides, phosphates, or sulfates, as well as the salts of organicacids such as acetates, proprionates, malonates, or benzoates.

Therapeutic compositions can also contain liquids, such as water,saline, glycerol, and ethanol, as well as substances such as wettingagents, emulsifying agents, or pH buffering agents. Liposomes, such asthose described in U.S. Pat. N o. 5,422,120, WO 95/13796, WO 91/14445,or EP 524,968 B1, can also be used as a carrier for the therapeuticcomposition.

Typically, a therapeutic composition is prepared as an injectable,either as a liquid solution or suspension, however, solid forms suitablefor solution in, or suspension in, liquid vehicles prior to injectioncan also be prepared. A composition can also be formulated into anenteric coated tablet or gel capsule according to known methods in theart, such as those described in U.S. Pat. No. 4,853,230, EP 225,189, AU9,224,296, and AU 9,230,801.

Administration of the therapeutic agents of the invention can includelocal or systemic administration, including injection, oraladministration, particle gun, or catheterized administration, andtopical administration. Various methods can be used to administer atherapeutic composition directly to a specific site in the body.

For treatment of tumors, for example, a small tumor or metastatic lesioncan be located and a therapeutic composition injected several times inseveral different locations within the body of the tumor. Alternatively,arteries which serve a tumor can be identified, and a therapeuticcomposition injected into such an artery, in order to deliver thecomposition directly into the tumor.

A tumor which has a necrotic center can be aspirated and the compositioninjected directly into the now empty center of the tumor. A therapeuticcomposition can be directly administered to the surface of a tumor, forexample, by topical application of the composition. X-ray imaging can beused to assist in certain of the above delivery methods. Combinationtherapeutic agents. including a protein or polypeptide or a subgenomicpolynucleotide and other therapeutic agents, can be administeredsimultaneously or sequentially.

Receptor-mediated targeted delivery can be used to deliver therapeuticcompositions containing subgenomic polynucleotides, proteins, orreagents such as antibodies, ribozymes, or antisense oligonucleotides ofthe invention to specific tissues. Receptor-mediated delivery techniquesare described in, for example, Findeis et al. (1993), Trends inBiotechnol. 11, 202-05; Chiou et al. (1994), GENE THERAPEUTICS. METHODSAND APPLICATIONS OF DIRECT GENE TRANSFER (J. A. Wolff, ed.); Wu & Wu(1988), J. Biol. Chem. 263, 621-24, Wu et al. (1994), J. Biol. Chem.269, 542-46; Zenke et al (1990), Proc. Natl. Acad. Sci. U.S.A.87:3655-59; Wu et al. (1991), J. Biol. Chem. 266, 338-42.

Alternatively, therapeutic compositions can be introduced into humancells ex vivo and the cells then replaced into the human. Cells can beremoved from a variety of locations including, for example, from aselected tumor or from an affected organ. In addition, a therapeuticcomposition can be inserted into non-affected, for example, dermalfibroblasts or peripheral blood leukocytes. If desired, particularfractions of cells such as a T cell subset or stem cells can also bespecifically removed from the blood (see, for example, PCT WO 91/16116).The removed cells can then be contacted with a therapeutic compositionutilizing any of the above-described techniques, followed by the returnof the cells to the human, preferably to or within the vicinity of atumor or other site to be treated. The methods described above canadditionally comprise the steps of depleting fibroblasts or othernon-contaminating tumor cells subsequent to removing tumor cells from ahuman, and/or the step of inactivating the cells, for example, byirradiation.

Both the dose of a composition and the means of administration can bedetermined based on the specific qualities of the therapeuticcomposition, the condition, age, and weight of the patient, theprogression of the disease, and other relevant factors. Preferably, atherapeutic composition of the invention increases or decreasesexpression of a polynucleotide by 50%, 60%, 70%, or 80%. Mostpreferably, expression of the polynucleotide is increased or decreasedby 90%, 95%, 99%, or 100%. The effectiveness of the mechanism chosen toalter expression of the polynucleotide can be assessed using methodswell known in the art, such as hybridization of nucleotide probes tomRNA of the polynucleotide, quantitative RT-PCR, or detection of aprotein or polypeptide using specific antibodies.

If the composition contains protein, polypeptide, or antibody, effectivedosages of the composition are in the range of about 5 μg to about 50μg/kg of patient body weight, about 50 μg to about 5 mg/kg, about 100 μgto about 500 μg/kg of patient body weight, and about 200 to about 250μkg.

Therapeutic compositions containing subgenomic polynucleotides can beadministered- in a range of about 100 ng to about 200 mg of DNA forlocal administration in a gene therapy protocol. Concentration ranges ofabout 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg toabout 500 μg, and about 20 μg to about 100 μg of DNA can also be usedduring a gene therapy protocol. Factors such as method of action andefficacy of transformation and expression are considerations that willeffect the dosage required for ultimate efficacy of the subgenomicpolynucleotides. Where greater expression is desired over a larger areaof tissue, larger amounts of subgenomic polynucleotides or the sameamounts re-administered in a successive protocol of administrations, orseveral administrations to different adjacent or close tissue portionsof, for example, a tumor site, may be required to effect a positivetherapeutic outcome. In all cases, routine experimentation in clinicaltrials will determine specific ranges for optimal therapeutic effect.

The therapeutic compositions are useful in treating pancreatic cancerand pancreatic dysplasia, as well as other types of cancers such as:bone cancer; brain tumors; breast cancer; endocrine system cancers, suchas cancers of the thyroid, pituitary, and adrenal glands and thepancreatic islets; gastrointestinal cancers, such as cancer of the anus,colon, esophagus, gallbladder, stomach, liver, and rectum; genitourinarycancers such as cancer of the penis, prostate and testes; gynecologicalcancers, such as cancer of the ovaries, cervix, endometrium, uterus,fallopian tubes, vagina, and vulva; head and neck cancers, such ashypopharyngeal, laryngeal, oropharyngeal cancers, lip, mouth and oralcancers, cancer of the salivary gland, cancer of the aerodigestive tractand sinus cancer, leukemia; lymphomas including Hodgkin's andnon-Hodgkin's lymphoma, metastatic cancer; myelomas; sarcomas; skincancer; urinary tract cancers including bladder, kidney and urethralcancers; and pediatric cancers, such as pediatric brain tumors,leukemia, lymphomas, sarcomas, liver cancer and neuroblastoma andretinoblastoma.

The following example provides data and experimental procedures.However, the invention is not limited to the example. The invention isdefined in the specification as a whole which includes the claims.

EXAMPLE 1

A family was identified that had several members who had been diagnosedwith pancreatic cancer. The pathological features of disease in thefamily included progression from normal to metaplasia to dysplasia tocancer. Tissues were obtained from a member of the family diagnosed withpancreatic cancer, from a member of the family diagnosed with dysplasiaof pancreatic cells, from a person unrelated to the family diagnosedwith pancreatitis, and from a person unrelated to the family with anormal pancreas.

Ductal cells from the tissues of each of these subjects were culturedand mRNA was isolated from the cultures. The mRNA was subjected toreverse transcriptase polymerase chain reaction using 200 primer pairs(10 anchored and 20 arbitrary primers). The resulting cDNA was subjectedto a differential display in which the cDNA from each of the 4 sampleswere compared on a gel. Bands of cDNA that appeared to be up-ordown-regulated in the dysplastic or pancreatic cancer samples, ascompared to the normal and pancreatitis samples, were cut from the gel,amplified, cloned, and sequenced.

The following polynucleotides sequences, as shown in SEQ ID NOS: 1-5,were identified as being mis-regulated in pancreatic cancer or dysplasiaor both:

TABLE 1 Up-Regulated and Down-Regulated Polynucleotides in PancreaticCancer and Dysplasia SEQ ID NO Regulation Status SEQ ID NO: 1 Up indysplasia only SEQ ID NO: 2 Up in dysplasia and cancer SEQ ID NO: 3 Upin dysplasia only SEQ ID NO: 4 Up in dysplasia only SEQ ID NO: 5 Up indysplasia and cancer SEQ ID NO: 6 Up in dysplasia only SEQ ID NO: 7 Upin dysplasia only SEQ ID NO: 8 Up in dysplasia only SEQ ID NO: 9 Up indysplasia only SEQ ID NO: 10 Up in dysplasia only SEQ ID NO: 11 Up indysplasia only SEQ ID NO: 12 Down in cancer only SEQ ID NO: 13 Up indysplasia only SEQ ID NO: 14 Up in dysplasia only SEQ ID NO: 15 Up indysplasia and cancer

All patents, published patent applications and publications cited hereinare incorporated by reference as if set forth fully herein.

Although certain preferred embodiments have been described herein, it isnot intended that such embodiments be construed as limitations on thescope of the invention except as set forth in the following claims.

1. A method of diagnosing cancer in a patient comprising comparing theamount of a polypeptide in a patient sample and in a normal sample,wherein said polypeptide comprises the polypeptide encoded by SEQ ID NO:15, or a naturally occurring variant of SEQ ID NO:15, wherein saidnaturally occurring variant hybridizes to SEQ ID NO:15 under conditionsincluding a wash in 2×SCC, 0.1% SDS at 50° C. for 20 minutes; andwherein a patient sample containing more of said polypeptide than thenormal sample is identified as cancerous.
 2. A method of diagnosingdysplasia in a patient comprising comparing the amount of a polypeptidein a patient sample and in a normal sample, wherein said polypeptidecomprises the polypeptide encoded by SEQ ID NO: 15, or a naturallyoccurring variant of SEQ ID NO:15, wherein said naturally occurringvariant hybridizes to SEQ ID NO:15 under conditions including a wash in2×SCC, 0.1% SDS at 50° C. for 20 minutes; and wherein a patient samplecontaining more of said polypeptide than the normal sample is identifiedas dysplastic.
 3. A method of diagnosing cancer in a patient comprisingdetermining the amount of a polypeptide in a patient sample and in anormal sample, wherein said polypeptide is encoded by a polynucleotideselected from the group consisting of SEQ ID NOS: 1, 3-4, 6-11, and13-14; determining the amount of a second polypeptide in the patientsample and in the normal sample, wherein said polypeptide is encoded bySEQ ID NO:15; and comparing the determined amounts; wherein a patientsample which contains more of the second polypeptide than the normalsample and which contains substantially the same amount of the firstpolypeptide, as compared to the normal sample, is identified ascancerous.
 4. The method of claim 1 or 3, wherein the cancer ispancreatic cancer.
 5. The method of claim 2, wherein the dysplasia ispancreatic dysplasia.
 6. The method of any one of claims 1-3, whereinthe patient sample and the normal sample comprise pancreatic cells.